The long-term objective in Project 2 is to gain understanding of the biochemical and biophysical mechanisms by which mutations from each MH/CCD hot spot alter key regulatory functions of the RyR1 Ca2+ channel complex, and how these changes deregulate SR Ca2+ transport in skeletal muscle SR, and dendritic cells that express MH/CCD RyR1. HYPOTHESIS I) MH/CCD mutations alter cytoplasmic and luminal regulation of RyR1 by ligands by changing the activation energy (Ea) needed for conformational transitions of the channel. A1.1. Define differences among 11 MH/CCD and wild type (Wt) genotypes on expression of key triadic proteins, SR loading capacity and their relationship to altered cation interactions at H- and Lsites using equilibrium [3H]ryanodine ([3H]Ry) binding analysis. A1.2. To analyze mechanisms responsible for altered regulation by cytoplasmic Ca2+, Mg2+, and ATP and luminal Ca2+ anc calsequestrin (CSQ) using selected RyR1 channels. A1.3. Compare the differences in activation energy (Ea) for selected MH/CCD mutations. A1.4. Analyze how halothane and dantrolene differentially influence the stability of closed and open states of selected MH/CCD RyR1s. HYPOTHESIS II) MH/CCD mutations disrupt the redox-sensing properties of RyR1. A2.1. To establish if differences in transmembrane redox regulation and GSH/GSSG transport contribute to the pathophysiology of MH/CCD. HYPOTHESIS III) MH/CCD mutations produce a dysfunctional phenotype in dendritic cells. A3.1. Elucidate how MH/CCD mutations alter murine dendritic cell activation, dendritic cell secretion of IL-6 mediated by ATP acting at P2Y receptors, and if and how MH/CCD mutations influence dendritic cell activation of T-cells. A3.2. To extend these studies to DCs cultured from human blood.